Repurposing the aldose reductase inhibitor and diabetic neuropathy drug epalrestat for the congenital disorder of glycosylation PMM2-CDG

Abstract

Phosphomannomutase 2 deficiency, or PMM2-CDG, is the most common congenital disorder of glycosylation affecting over 1,000 patients globally. There are no approved drugs that treat the symptoms or root cause of PMM2-CDG. In order to identify clinically actionable compounds that boost human PMM2 enzyme function, we performed a multi-species drug repurposing screen using a first-ever worm model of PMM2-CDG followed by PMM2 enzyme functional studies in PMM2-CDG patient fibroblasts.Drug repurposing candidates from this study, and drug repurposing candidates from a previously published study using yeast models of PMM2-CDG, were tested for their effect on human PMM2 enzyme activity in PMM2-CDG fibroblasts. Of the 20 repurposing candidates discovered in the wormbased phenotypic screen, 12 are plant-based polyphenols. Insights from structure-activity relationships revealed epalrestat, the only antidiabetic aldose reductase inhibitor approved for use in humans, as a firstin-class PMM2 enzyme activator. Epalrestat increased PMM2 enzymatic activity in four PMM2-CDG patient fibroblast lines with genotypes R141H/F119L, R141H/E139K, R141H/N216I and R141H/F183S. PMM2 enzyme activity gains range from 30% to 400% over baseline depending on genotype.Pharmacological inhibition of aldose reductase by epalrestat may shunt glucose from the polyol pathway to glucose-1,6-bisphosphate, which is an endogenous stabilizer and coactivator of PMM2 homodimerization. Epalrestat is a safe, oral and brain penetrant drug that was approved 27 years ago in Japan to treat diabetic neuropathy in geriatric populations. We demonstrate that epalrestat is the first small molecule activator of PMM2 enzyme activity with the potential to treat peripheral neuropathy and correct the underlying enzyme deficiency in a majority of pediatric and adult PMM2-CDG patients.Keywords: Phosphomannomutase 2 deficiency, drug repurposing, PMM2-CDG, congenital disorder of glycosylation, aldose reductase inhibitor, epalrestat conserved process that occurs in all animal cells throughout development and adulthood (Chang et al, 2018). PMM2-CDG is a multi-system, multi-organ disease because a minimal level of glycosylation is required at all times in all cells of the body, with cell types or organs more or less vulnerable to the complex sequelae of hypoglycosylation. Although a clear genotype-phenotype relationship is obscured by genetic and possibly environmental modifiers, as the residual level of PMM2 enzymatic activity increases the number and severity of organ systems affected decreases. For example, patients homozygous for a mutation in the promoter of PMM2 do not get PMM2-CDG or even a mild form of PMM2-CDG but instead have hyperinsulinemic hypoglycemia and polycystic kidney disease because this mutation impairs binding by a kidney-and pancreas-specific transcription factor to a chromatin loop in the promoter of PMM2 (Cabezas et al, 2017). As another example, hypoglycosylation of the calcium channel CACNA1A caused a gain-of-function channelopathy that in turn leads to an i...

β-Glucose-1,6-Bisphosphate Stabilizes Pathological Phophomannomutase2 Mutants In Vitro and Represents a Lead Compound to Develop Pharmacological Chaperones for the Most Common Disorder of Glycosylation, PMM2-CDG

“…None of these molecules resembles natural ligands of PMM2. α -cyano-4-hydroxycinnamic acid enhanced PMM2 enzymatic activity in fibroblasts derived from patients and in a nematode model of PMM2-CDG [25]. Since α -cyano-4-hydroxycinnamic acid shares the carboxylic acid-containing pharmacophore of aldose reductase inhibitors, Perlestein and co-workers [25] tested other commercially available inhibitors of the same enzyme in the nematode model and fibroblasts.…”

“…α -cyano-4-hydroxycinnamic acid enhanced PMM2 enzymatic activity in fibroblasts derived from patients and in a nematode model of PMM2-CDG [25]. Since α -cyano-4-hydroxycinnamic acid shares the carboxylic acid-containing pharmacophore of aldose reductase inhibitors, Perlestein and co-workers [25] tested other commercially available inhibitors of the same enzyme in the nematode model and fibroblasts. They found that epalrestat, which is a safe, orally bioavailable, and brain penetrant aldose reductase inhibitor used to treat diabetic peripheral neuropathy, rescued PMM2 enzymatic activity in both species.…”

“…Epalrest is a monocarboxylic acid and contains a phenyl and a rhodanine group and does not resemble chemically PMM2 ligand. Direct binding and stabilization to mutant PMM2 proteins is not among the modes of actions proposed by Perlestein and co-workers for epalrest [25].…”

Multi-Species Phenotypic Screening across Disease Models of Mucolipidosis Type IV

“…Luckily the manual scoring format provide a z′-score of >1.5 with 41 hits that replicated in more than two experiments ( SI Figure 2.3 ). The low hit discovery rate in our fly screen may not be surprising as drug exposure related artifacts such as reduced feeding relating to the preference of flies for each compound, uncertainty about the amount of food consumed, compound stability in fly food, stochasticity due to small numbers of animals per well, and tissue concentration achieved by each compound are known (Qi, et al, 2015; Ja, et al, 2007; Marx, 2015; Iyer, et al, 2019). To enable our ability to compare the fly hits with the orthogonal screening efforts in worms and fibroblasts, we chose to be more inclusive of our hits for analysis.…”

Spontaneous improvement of carbohydrate-deficient transferrin in PMM2-CDG without mannose observed in CDG natural history study

“…We are concerned about the futility of spending time and effort to pursue additional studies of mannose in PMM2-CDG, where there is very little evidence for efficacy. We note that there have been a number of recent advances in approaches to treat PMM2-CDG [13], including: a recently reported open-label study demonstrating improvement in ataxia with acetazolamide [14], pharmacologic chaperones [15] or aldose reductase inhibitors identified through a drug-repurposing screen [16], and mannose-1-phosphate replacement therapies intended to bypass the PMM2-dependant biosynthetic step [13].…”